CN114891712B - Recombinant escherichia coli for improving yield of N-acetylneuraminic acid - Google Patents

Recombinant escherichia coli for improving yield of N-acetylneuraminic acid Download PDF

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CN114891712B
CN114891712B CN202210690425.4A CN202210690425A CN114891712B CN 114891712 B CN114891712 B CN 114891712B CN 202210690425 A CN202210690425 A CN 202210690425A CN 114891712 B CN114891712 B CN 114891712B
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CN114891712A (en
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刘延峰
刘龙
刘畅
吕雪芹
田荣臻
李江华
堵国成
陈坚
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Jiangnan University
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Abstract

The invention discloses recombinant escherichia coli for improving the yield of N-acetylneuraminic acid and a construction method thereof, belonging to the technical fields of metabolic engineering and genetic engineering. The invention constructs recombinant strain BL21 (DE 3) delta nanATED, delta nagABE, delta manXYZ with N-acetylneuraminic acid synthesis capability, wherein Pgpre-NeuC, delta poxB are expressed in a genome in a recombinant way, and the low-strength constitutive promoter P is expressed in a recombinant way grpE Regulated glmM, consisting of the medium strength constitutive promoter P ssrA Regulatory glmU and glmS mutant glmS A And using low-strength constitutive promoter P grpE The expression of NemNaeuB from Neisseria meningitidis is regulated, a recombinant strain with improved N-acetylneuraminic acid yield is obtained, the yield reaches 11.28g/L, and a foundation is laid for the industrialized production of N-acetylneuraminic acid.

Description

Recombinant escherichia coli for improving yield of N-acetylneuraminic acid
Technical Field
The invention relates to a recombinant escherichia coli for improving the yield of N-acetylneuraminic acid, belonging to the technical fields of metabolic engineering and genetic engineering.
Background
N-acetylneuraminic acid (N-acetylneuraminic acid) widely exists in the nature, has very important application value, can be used for resisting bacteria and expelling toxin in medical and health care aspects, can be used for developing sialidase inhibitor type anti-influenza drugs as a precursor of anti-influenza virus drug zanamivir, and can be used for improving the antibacterial capability of intestinal tracts when being added into foods, wherein the N-acetylneuraminic acid is a conditionally essential nutritional factor for the growth and development of the brain of infants, can promote the memory and intelligence development.
Coli (Escherichia coli) is a model industrial microorganism which is applied to metabolic engineering in large scale to produce chemicals, and has very wide application in medicine, chemical industry, agriculture and the like. In recent years, with the research and development of synthetic biology, the gene editing tools of escherichia coli are various, simple to operate and mature. In addition, the escherichia coli has the advantages of easy culture, clear genetic background, rapid growth, high expression, simple genetic operation, easy metabolic engineering transformation and the like.
In development and transformation of chassis cells, plasmid free expression exogenous genes are poor in stability, metabolic burden of cells is easily increased, and accordingly yield and production efficiency of target products are reduced, large-scale industrial production is difficult to meet, and stable expression can be achieved by integrating exogenous genes into escherichia coli genome. At present, the metabolic pathway in the high-yield strain of N-acetylneuraminic acid mainly uses glucose as a substrate, and N-acetylglucosamine is used as a precursor to express AGE enzyme in vitro, which is easy to cause insufficient metabolic flux intensity, so that the feedback inhibition effect in the speed limiting step is relieved, the metabolic flux of the synthetic pathway of N-acetylneuraminic acid is strengthened, and it is important to find exogenous enzyme, a fermentation carbon source and an efficient synthetic pathway which are suitable for the production of N-acetylneuraminic acid in escherichia coli.
Disclosure of Invention
The invention aims to provide a recombinant escherichia coli strain for improving the yield of N-acetylneuraminic acid and a construction method thereof.
The invention provides a recombinant escherichia coli, which knocks out a Gene N-acetylglucosamine-6-phosphate deacetylase nagA (Gene ID: 945289), glucosamine-6-phosphate deaminase nagB (Gene ID: 945290), N-acetylglucosamine-specific EIICBA component nagE (Gene ID: 945292), N-acetylneuraminic acid transporter-related Gene N-acetylneuraminic acid lyase nanA (Gene ID: 947742), sialic acid transporter nanT (Gene ID: 947740), N-acetylmannosamine-6-phosphate 2-epimerase nanE (Gene ID: 947745), N-acetylmannosamine kinase nanK (Gene ID: 947757), mannose-specific EIIAB component-related Gene manXYZ ((Gene ID:946334,946332)) pyruvate oxidase poxB Gene (Gene ID: 946132), and expression of endogenous enzyme glmM (Gene ID: 947692) regulated by a low-strength constitutive promoter, glmU (Gene ID: 948246) regulated by a medium-strength constitutive promoter, and glucosamine synthase mutant glmS A And (Neisseria meningitidis) source N-acetylneuraminic acid synthase NemNaeuB and Neisseria meningitidis (Neisseria meningitidis) source UDP-N-acetylglucosamine-2-epimerase NeuC were introduced.
In one embodiment, the endogenous enzymes glmM, glmU and glucosamine synthase mutant glmS under the control of the constitutive promoter A And N-acetylneuraminic acid synthase NemNauB are both expressed integrally on the genome.
In one embodiment, glmM, glmU and the glucosamine synthase mutant glmS A The integration site of (a) is the site of the motA Gene of E.coli (Gene ID: 947564).
In one embodiment, the neminub is integrated at the locus of the Δpoxb gene.
In one embodiment, the low-intensity groupThe shaping promoter is P grpE The medium-strength constitutive promoter is P ssrA 。
In one embodiment, the N-acetylneuraminic acid synthase NemNauB is passed through promoter P trc Regulating and controlling expression; the UDP-N-acetylglucosamine-2-epimerase NeuC passes through the promoter P grpE Regulating and controlling expression.
In one embodiment, E.coli.BL21 (DE 3) ΔnanATEK, ΔnagABE, ΔmanXYZ, ΔpoxB are used as starting strain, and P is integrated at ΔmanXYZ grpE Regulated endogenous enzyme NeuC gene and integration of P at ΔpoxB grpE Regulated nemineup b.
In one embodiment, the amino acid sequence of the UDP-N-acetamido-2-epimerase NeuC is shown in SEQ ID NO. 1; the nucleotide sequence of the coding NeuC gene is shown as SEQ ID NO. 2.
In one embodiment, the amino acid sequence of N-acetylneuraminic acid synthase NeuB is shown in SEQ ID NO.3, SEQ ID NO.16 or SEQ ID NO. 18; the nucleotide sequence of the coding NeuB gene is shown as SEQ ID NO.4, SEQ ID NO.17 or SEQ ID NO.19 respectively.
In one embodiment, the glucosamine synthase mutant glmS A The amino acid sequence of (2) is shown as SEQ ID NO. 5; encoding glmS A The nucleotide sequence of the gene is shown as SEQ ID NO. 6.
In one embodiment, the glmM, glmU-glmS is expressed using a combination of promoters of different strengths A The method comprises the steps of carrying out a first treatment on the surface of the The promoters of different strengths are selected from P ssrA 、P grpE 、P 224 。
In one embodiment, the promoter P shown in SEQ ID NO.10 is used grpE The glmM is expressed in an intensified manner by using a promoter P shown in SEQ ID NO.8 ssrA Enhanced expression of the glmU and mutant glmS A 。
In one embodiment, the promoter P shown in SEQ ID NO.10 is used grpE And (3) enhancing the expression of the N-acetylneuraminic acid synthase NemNauB.
The invention also provides a method for improving the N-acetylnerve of the recombinant escherichia coliA method for synthesizing amino acid, which comprises the steps of using recombinant E.coli.BL21 (DE 3) ΔnanATEK, ΔnagABE, ΔmanXYZ:: P gprE NeuC, ΔpoxB as starting strain, glmM, glmU and glmS mutant glmS were enriched with a combination of constitutive promoters of different intensities A And optimizing the expression level of NeuB derived from Neisseria meningitidis (Neisseria meningitidis) on the genome with promoters of different strengths, selected from the promoters shown in any one of SEQ ID NOS.7 to 15.
In one embodiment, a promoter P is used grpE Regulating glmM gene and using P ssrA Regulatory glmU and glmS mutant glmS A Is expressed in terms of the expression of (a),
in one embodiment, a promoter P is used grpE Regulate the expression of N-acetylneuraminic acid synthase NemNauB and NeuC.
The invention also provides application of the recombinant escherichia coli in fermentation production of N-acetylneuraminic acid.
In one embodiment, the fermentation uses glycerol as a carbon source, and the concentration of glycerol can be selected from 20-40 g/L.
In one embodiment, the fermentation is performed in a medium containing 30g/L glycerol, 6g/L urea, 3.8mg/L zinc sulfate heptahydrate, 0.33g/L manganese sulfate monohydrate, 5g/L iron sulfate heptahydrate, 0.1g/L copper sulfate pentahydrate, 0.1g/L cobalt chloride hexahydrate, 4.8g/L yeast powder, 2.4g/L tryptone, 5.336g/L potassium dihydrogen phosphate, 3.284g/L dipotassium phosphate trihydrate, 2.84g/L citric acid monohydrate, 2g/L magnesium sulfate heptahydrate, 4g/L ammonium sulfate, and pH adjusted to 7.
In one embodiment, the method inoculates the recombinant escherichia coli after genetic modification into LB culture medium, cultures for 12-14 hours at 37 ℃ to obtain seed solution, and then transfers the seed solution into fermentation medium for fermentation with 1-2% of inoculum size.
In one embodiment, the fermentation is carried out at 35-37 ℃ for at least 24 hours.
The invention provides application of the escherichia coli in producing a product containing N-acetylneuraminic acid.
In one embodiment, the product includes, but is not limited to, a pharmaceutical or nutraceutical product
The beneficial effects are that: the recombinant escherichia coli provided by the invention can realize extracellular accumulation of N-acetylneuraminic acid, and glmM, glmU and glmS are reinforced by adopting constitutive escherichia coli promoters with different intensities A The metabolic flux of the N-acetylneuraminic acid synthesis pathway is improved, and the expression level of N-acetylneuraminic acid synthase NeuB of different sources on the genome is optimized by using constitutive promoters with different intensities, so that the yield of the N-acetylneuraminic acid is improved to more than 11.28g/L when the constructed recombinant escherichia coli is fermented for 48 hours.
Drawings
FIG. 1 is a metabolic scheme of N-acetylneuraminic acid.
Detailed Description
Glucosamine synthase mutant glmS A The nucleotide sequence of the gene is shown as SEQ ID NO. 6. The nucleotide sequence of the Neisseria meningitidis-derived N-acetylneuraminic acid synthase NemNaB is shown in SEQ ID NO. 17. The nucleotide sequence of the Moritella viscosa-derived N-acetylneuraminic acid synthase encoding gene MovNeuB is shown in SEQ ID NO. 4. The nucleotide sequence of the EcoNeuB gene coded by the Ecoli-derived N-acetylneuraminic acid synthase is shown in SEQ ID NO. 19.
Promoter P trc 、P ssrA 、P dnakj 、P grpE 、P 566 、P 224 、P 333 、P tac 、P alsAR The nucleotide sequences of (2) are SEQ ID NO. 7-15 respectively;
culturing and fermenting recombinant escherichia coli seeds:
seed liquid culture medium: 10g/L tryptone, 10g/L sodium chloride, 5g/L yeast powder.
The formula of the fermentation medium is as follows: 30g/L glycerin, 6g/L urea, 3.8mg/L zinc sulfate heptahydrate, 0.33g/L manganese sulfate monohydrate, 5g/L iron sulfate heptahydrate, 0.1g/L copper sulfate pentahydrate, 0.1g/L cobalt chloride hexahydrate, 4.8g/L yeast powder, 2.4g/L tryptone, 5.336g/L potassium dihydrogen phosphate, 3.284g/L dipotassium phosphate trihydrate, 2.84g/L citric acid monohydrate, 2g/L magnesium sulfate heptahydrate, 4g/L ammonium sulfate, pH adjusted to 7
Culture conditions: inoculating recombinant escherichia coli into LB culture medium, culturing at 37 ℃ for 12-14h at 220rpm to obtain seed liquid, transferring 1-2% of the seed liquid into fermentation culture medium for fermentation, and reacting at 37 ℃ for 48h at 220 rpm.
The sample detection method comprises the following steps: the detection of N-acetylneuraminic acid is carried out by Agilent liquid chromatography, the chromatographic column is Aminex HPX-87H column (300X 7.8 mM), the ultraviolet 210nm detection absorption peak, the mobile phase is 10mM sulfuric acid, the flow rate is 0.5mL/min, and the peak outlet time of N-acetylneuraminic acid is about 9.8 minutes.
EXAMPLE 1 recombinant strains Ecoli.BL21 (DE 3) ΔnanATEK, ΔnagABE, ΔmanXYZ:: P gprE -NeuC,ΔpoxB::P trc Construction of MovNeuB
(1) Construction of nagABE, nanATEK, manXYZ and poxB knockout Strain
The knockdown of the glucosamine transport related phosphotransferase system related Gene N-acetylglucosamine-6-phosphodeacetylase nagA (Gene ID: 945289), glucosamine-6-phosphodeaminase nagB (Gene ID: 945290), PTS system N-acetylglucosamine specific EIICBA component nagE (Gene ID: 945292), N-acetylneuraminic acid transport vector related Gene N-acetylneuraminic acid lyase nanA (Gene ID: 947742), sialic acid transporter nanT (Gene ID: 947740), N-acetylmannosamine-6-phospho2-epimerase nanE (Gene ID: 947745), N-acetylmannosamine kinase nanK (Gene ID: 947757), PTS system mannose specific EIIAB component related Gene manXYZ (Gene ID:946334,946332) pyruvate oxidase poB Gene (Gene ID: 946132) on the genome of E.coli was achieved by CRISPER/Cas9 Gene editing technology. Wherein nagABE has an N20 sequence of attgccctgagcaaggagcc, nanATEK has an N20 sequence of gctttggtatgaaaattgta, manXYZ has an N20 sequence of acgaagccgaggtagaagaa, poxB has an N20 sequence of ggtgaaaatagcgtcatcgg. Firstly, pCas9 plasmid containing Cas9 cutting protein is transformed into escherichia coli host bacteria by a chemical transformation method, escherichia coli BL21 (DE 3) is used as a template, the upstream and downstream fragments of a knocking-out site are amplified by PCR to obtain fragments by about 1000bp respectively, and the fragments are connected with a targeting cutting plasmid pTarget carrier in a CRISPER/Cas9 system by a Gibson assembly kit. After the plasmid is constructed and sequenced, the competent cells of the escherichia coli containing pCas9 are prepared, so that the target gene is knocked out. After transformation, the transformants were plated on resistant plates, single colonies were picked for colony PCR to verify positive transformants and sequenced, recombinant e.coli with nagABE, nanATEK, manXYZ and poxB knocked out on the genome was constructed in sequence, and the strain was designated as NBC-1.
(2) Construction of genomic recombinant integration NeuC fragments
Synthesizing a nucleotide sequence of a coding gene NeuC (shown as SEQ ID NO. 2) of UDP-N-acetylglucosamine-2-epimerase NeuC (the amino acid sequence of which is shown as SEQ ID NO. 1) from Neisseria meningitidis; the integration site of NeuC was selected for knock-in at the site of the mannose-specific EIIAB component-related gene manXYZ of the PTS system.
E.coli BL21 (DE 3) is used as a template, primers NeuC-L-F1 and NeuC-L-R1 are designed, and a NeuC left arm gene fragment is amplified, recombined and integrated;
NeuC-L-F1：5’-catcaataccgtttccggcaaaggc-3’,
NeuC-L-R1：
5’-CTCCCGGACCAAAACGAAAAAAGACGCTTTTCAGCGTCTTTTTTTTTTTTTTTGGTAC CGAGgaatctgttagaggcgcaatagtgacag-3’,
p with synthetic nucleotide sequence shown as SEQ ID NO.10 grpE A promoter fragment;
e.coli BL21 (DE 3) is used as a template, primers NeuC-R-F1 and NeuC-R-R1 are designed, and a recombinant integration NeuC right arm gene fragment is amplified;
NeuC-R-F1:5’-CGGGGGCTTTctcatgcgtttcccaggtggaagccctatttcttttatg-3’；
NeuC-R-R1:5’-gtagagttcactcctgccgatccg-3’
amplifying the amplified NeuC left arm gene fragment and P grpE The promoter fragment, the NeuC gene fragment and the NeuC right arm fragment are constructed into a recombinant integrated NeuC gene fragment P by fusion PCR technology grpE -NeuC。
(3) Construction of genomic recombinant integration NeuB fragment
Synthesizing a nucleotide sequence (shown as SEQ ID NO. 4) of an encoding gene NeuB of the N-acetylneuraminic acid synthase encoding gene NeuB (the amino acid sequence is shown as SEQ ID NO. 3) from Moritella viscosa sources; the integration site of NeuB was selected for knock-in integration at the site of the pyruvate oxidase poxB Gene (Gene ID: 946132).
E.coli BL21 (DE 3) is used as a template, primers NeuB-L-F1 and NeuB-L-R1 are designed, and a NeuB left arm gene fragment is amplified, recombined and integrated;
NeuB-L-F1：5’-gaggcgttaatcagcacgtttctcgct-3’,
NeuB-L-R1：
5’-CACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAaaagggtggcatttcccgtcataataa ggacat-3’
synthetic nucleotide sequence is shown as P shown in SEQ ID NO.14 trc A promoter fragment;
e.coli BL21 (DE 3) is used as a template, primers NeuB-R-F1 and NeuB-R-R1 are designed, and a recombinant integration NeuB right arm gene fragment is amplified;
NeuB-R-F1：
5’-GCCGAAGCGGGTTTTTACGTAAAACAGGTGAAACTGACggttctccatctcctgaatgtgataacggtaacaagtt-3’,
NeuB-R-R1：5’-aatatgcactggtcagcgtgcgtaactc-3’
amplifying the amplified NeuB left arm gene fragment and P trc The promoter fragment, the NeuB gene fragment and the NeuB right arm fragment are constructed into a recombinant integrated NeuB gene fragment P by fusion PCR technology trc -NeuB。
(4) Integration of genes
Fusion fragment P constructed in step (2) grpE After NeuC is connected with a targeting cleavage plasmid pTarget vector containing an N20 recognition sequence (agccctttctttttatagtt) in a CRISPER/Cas9 system through a Gibson assembly kit, the constructed plasmid is transformed into E.coli NBC-1 competent cells constructed in the step (1) containing the Cas9 plasmid by adopting a chemical transformation method, so that P is obtained gprE Recombinant strain of NeuC integrated at ΔmanXYZ, named NBC-2 after verification of correct.
Fusion fragment P of the fusion fragment constructed in the step (3) trc After the NeuB is connected with a targeting cleavage plasmid pTarget vector containing an N20 recognition sequence (ggtgaaaatagcgtcatcgg) in a CRISPER/Cas9 system through a Gibson assembly kitTransforming the constructed plasmid into competent cells of Escherichia coli NBC-2 by chemical transformation method to obtain P trc Recombinant strain of NeuB integrated at Δpoxb. Colony PCR verification was performed on the transformants, and the strain after successful integration was designated NBC-3.
Example 2 construction of glmM, glmU and glmS under the control of a combination of promoters of different strengths A Recombinant fragments
The genome of Escherichia coli BL21 (DE 3) is used as a template, primers glm-L-F2 and glm-L-R2 are designed, and recombinant P is amplified First promoter -glmM-P Second promoter -glmU-glmS A A left homology arm gene fragment of the integration site;
glm-L-F2:5’-acttagttAGCTTGGCCgtaacgcccgcagtttcggatc-3’；
glm-L-R2:5’-cctcggcattttattggcttacgg-3’；
the genome of Escherichia coli BL21 (DE 3) is used as a template, primers glm-R-F2 and glm-R-R2 are designed, and recombinant P is amplified First promoter -glmM-P Second promoter -glmU-glmS A A right homologous arm gene fragment of the integration site;
glm-R-F2：5’-GGTACCGAGgatttcgccatcaaccgataaagcagagc-3’；
glm-R-R2：5’-taaccttgaacagtgcccacaagcag-3’；
synthesis of glmS represented by the nucleotide sequence of SEQ ID No.6 A Fragments;
synthesizing promoter fragments with nucleotide sequences shown in SEQ ID NO.8, 9 and 12;
the genome of escherichia coli BL21 (DE 3) is used as a template, primers glmM-F2 and glmM R2 are designed, and a gene fragment of the recombinant integrated glmM is amplified;
glmM-F2:5’-ccctgaaactgatccccataataagcgaagttagcgagatgaatgcgaaaaaaacgGTTCACACAGGAAACCTATAATGatgagtaatcg-3’；
glmM-R2:5’-agatcccggtgcctaatgagtgag-3’；
the genome of escherichia coli BL21 (DE 3) is used as a template, primers glmU-F2 and glmU R2 are designed, and a gene fragment of recombinant integrated glmU is amplified;
glmU-F2:5’-cattgaggctggtcatggcgctcataaatctggtatacttacctttacacattGTTCACACAGGAAACCTATAATGatgttgaataatgc-3’；
glmU-R2:5’-ggatttctgctacatcacgttgcgc-3’；
obtaining the homology arm at the left side of the integration site, the promoter fragment (shown as SEQ ID NO.8, 9, 12, respectively), the glmM fragment, the glmU fragment, the glmS by PCR amplification A Fragment and right homology arm. Fusion PCR technique was used to separate the three-strength promoter fragments from the glmM fragment and glmU-S fragment A The fragments are fused, and the fragments regulated and controlled by the fragments are respectively named as glmM 1-glmM 3 according to different promoters; glmU-S A 1～glmU-S A 3. Wherein glmM1 corresponds to a polypeptide comprising P represented by SEQ ID NO.10 grpE A glmM fusion fragment of the promoter, glmM2 corresponding to the fragment containing P shown in SEQ ID NO.8 ssrA A glmM fusion fragment of the promoter, glmM3 corresponding to the fragment containing P shown in SEQ ID NO.12 224 glmM fusion fragment of promoter. glmU-S A Naming is similar, glmU-S A 1 corresponds to the sequence containing P shown in SEQ ID NO.10 grpE glmU-S of promoter A Fusion fragment, glmU-S A 2 corresponds to the sequence containing P shown in SEQ ID NO.8 ssrA glmU-S of promoter A Fusion fragment, glmU-S A 3 correspond to the sequence containing P shown in SEQ ID NO.12 224 glmU-S of promoter A Fusion fragments.
TABLE 1 promoter sequences
Example 3 control of glmM, glmU and glmS by combination of promoters of different strengths A Construction of recombinant strains of (2)
The recombinant E.coli host NBC-3 (E.coli.BL21 (DE 3) ΔnanATEK, ΔnagABE, ΔmanXYZ:: pgprE-NeuC, ΔpoxB::: ptrc-MovNeuB) constructed in example 1 was used as starting strain, and the glmM1 to glmM3 and glmU-S constructed in example 2 containing combinations of promoters of different intensities A 1～glmU-S A 3 through Gibson assembly kit and CRISPER/Cas9 systemN23 recognition sequence (tcgtcgattatctgcgcctgatt) targeting cleavage plasmid pTarget vector ligation and ligation of glmM and glmU-S A The combination of 9 different intensities was performed according to the requirements of the different intensities. After plasmid construction was sequenced, E.coli.BL21 (DE 3) ΔnanATEK, ΔnagABE:: glmSA, ΔmanXYZ:: pgprE-NeuC, ΔpoxB:: ptrc-MovNeuB E.coli host competent cells were prepared containing pCas9 to achieve glmM1 to glmM3 and glmU-S A 1～glmU-S A 3 recombinant integration at motA in the host bacterium. After transformation, transformants were plated on corresponding resistance plates, single colonies were picked for colony PCR to verify positive transformants and sequencing was performed again. Finally, the mixture contains glmM 1-glmM 3 and glmU-S A 1～glmU-S A 3 recombinant E.coli, designated NBC-5-1 to NBC-5-9, respectively (wherein NBC-5-1 corresponds to glmM 1-glmU-S) A 1, NBC-5-2 corresponds to glmM1-glmU-S A 2, NBC-5-3 corresponds to glmM1-glmU-S A 3, NBC-5-4 corresponds to glmM2-glmU-S A 1, NBC-5-5 corresponds to glmM2-glmU-S A 2, NBC-5-6 corresponds to glmM2-glmU-S A 3, NBC-5-7 corresponds to glmM3-glmU-S A 1, NBC-5-8 corresponds to glmM3-glmU-S A 2, NBC-5-9 corresponds to glmM3-glmU-S A 3)。
Recombinant escherichia coli NBC-5-1 to NBC-5-9 are inoculated in LB liquid culture medium respectively for overnight culture for 12-14 hours, and then seed solution is inoculated in TMM fermentation culture medium according to the inoculation amount of 2 percent for fermentation culture for 48 hours at 37 ℃ and 220 rpm. The yields of N-acetyl-D-aminomannose (ManNAc), a key precursor of N-acetylneuraminic acid (NeuAc), in the fermentation broth were determined to be 2.79g/L,9.24g/L,8.5g/L,2.75g/L,1.91g/L,2.72g/L,2.75g/L,2.72g/L,8.3g/L, respectively. E.coli engineering bacteria (i.e., P) with 9.24g/L yield of N-acetyl-D-amino mannose (ManNAc) grpE -glmM-P ssrA -glmU-glmS A ) Designated NBC-5.
EXAMPLE 4 construction of genomic integrated NeuB recombinant fragments
Synthesizing nucleotide sequences (shown as SEQ ID NO.4, 18 and 20) of the coding gene NeuB according to the N-acetylneuraminic acid synthase coding genes NeuB of different sources;
taking NemNeuB (amino acid sequence shown in SEQ ID NO. 17) derived from Neisseria meningitidis (Neisseria meningitidis) as an example: e.coli BL21 (DE 3) is used as a template, primers NemNauB-L-F1 and NemNauB-L-R1 are designed, and a recombinant integrated NemNauB left-arm gene fragment is amplified;
NemNeuB-L-F1：5’-gaggcgttaatcagcacgtttctcgct-3’；
NemNeuB-L-R1：5’-aaagggtggcatttcccgtcataataaggacat-3’；
synthesizing promoter fragments shown in nucleotide sequences of SEQ ID NO. 9-13 and SEQ ID NO. 15;
e.coli BL21 (DE 3) is used as a template, primers NemNauB-R-F1 and NemNauB-R-R1 are designed, and a recombinant integrated NemNauB right arm gene fragment is amplified;
NemNeuB-R-F1:5’-GCCGAAGCGGGTTTTTACGTAAAACAGGTGAAACTGACggttctccat ctcctgaatgtgataacggtaacaagtt-3’；
NemNeuB-R-R1:5’-aatatgcactggtcagcgtgcgtaactc-3’；
the amplified NemNanuB left arm gene fragment, promoter fragment (shown as SEQ ID NO. 8-12 and SEQ ID NO.15 respectively), nemNanuB gene fragment (nucleotide sequence shown as SEQ ID NO. 17) and NemNanuB right arm fragment are constructed into recombinant integrated NemNanuB gene fragment by fusion PCR technology, and are named as NemNanuB 1-NemNanuB 6 respectively according to different promoters; wherein NemNauB 1 correspondingly contains P shown in SEQ ID NO.8 ssrA NemNauB fusion fragment of promoter, nemNauB 2 corresponding to the promoter containing P shown in SEQ ID NO.10 grpE NemNauB fusion fragment of promoter, nemNauB 3 corresponding to the promoter containing P shown in SEQ ID NO.12 224 NemNauB fusion fragment of promoter, nemNauB 4 correspondingly contains P shown in SEQ ID NO.9 dnakj NemNauB fusion fragment of promoter, nemNauB 5 corresponding to the promoter containing P shown in SEQ ID NO.11 566 NemNauB fusion fragment of promoter, nemNauB 6 corresponding to the promoter containing P shown in SEQ ID NO.15 alsAR NemNaB fusion fragment of promoter.
According to the same strategy, recombinant integration fragments of MovNeuB (with a nucleotide sequence shown as SEQ ID NO. 4) from Moritella viscosa and EcoNeuB (with a nucleotide sequence shown as SEQ ID NO. 19) from E.coli are constructed, and the recombinant fragments are named as MovNeuB 1-MovNeuB 5 and EcoNeuB 1-EcoNeuB 6 respectively according to different promoters. Wherein MovNeuB 1-MovNeuB 5 respectively correspond to fusion fragments containing promoters shown by SEQ ID NO. 13-SEQ ID NO.15 and SEQ ID NO. 7; ecoNeuB 1-EcoNeuB 6 correspond to EcoNeuB fusion fragments containing SEQ ID NO. 7-10, 13, 15, respectively, the promoters shown.
TABLE 2 promoter sequences
EXAMPLE 5 construction of E.coli recombinant integration of NemNauB Gene
The NemNaeuB fragments (NemNaeuB 1-NemNaeuB 6) obtained in example 4 and regulated by promoters with different intensities are respectively connected with a targeting cleavage plasmid pTarget vector containing an N20 recognition sequence in a CRISPER/Cas9 system through a Gibson assembly kit, and then the constructed plasmid is transformed into NBC-5 escherichia coli competent cells containing Cas9 plasmid by a chemical transformation method, so that the original Ptrc-MovNeuB is replaced by the different promoters-NemNaeuB fragments. Positive transformants which were correctly sequenced and contained the DNA fragments of NemNauB regulated by promoters of different intensities were designated NBC8-1 to NBC8-6, respectively.
After the tool plasmid in the gene editing system was eliminated, the concentration of the product in the supernatant was measured by high performance liquid chromatography after culturing at 37℃and 220rpm for 48 hours in a fermentation medium, and the N-acetylneuraminic acid (NeuAc) yields detected in NBC8-1 to NBC8-6 were 8.76g/L,11.28g/L,5.2g/L,0.945g/L,9.87g/L and 2.575g/L, respectively. The results showed that NemNaeuB from Neisseria meningitidis (Neisseria meningitidis) was subjected to a low-strength promoter P grpE The regulated strain had a relatively highest yield of N-acetylneuraminic acid (NeuAc) of 11.28g/L and was designated NBC-8.
Comparative example 1
The large intestine of example 5 was weightedNemNeuB sequence (SEQ ID NO. 17) derived from Neisseria meningitidis (Neisseria meningitidis) in the group strain is replaced by MovNeuB (SEQ ID NO. 4) derived from Moritella viscosa and EcoNeuB (SEQ ID NO. 19) sequences derived from Ecoli respectively, and positive transformants which are correctly sequenced and contain DNA fragments of EcoNeuB, movNeuB regulated by promoters with different intensities are named NBC6-1 to NBC6-6 (respectively fused with P) tac 、P ssra 、P dnakj 、P grpE 、P alsaR 、P 333 Promoters), NBC7-1 to NBC7-5 (P fused to each other) tac 、P ssra 、P dnakj 、P 566 、P 333 、P trc A promoter). N-acetylneuraminic acid (NeuAc) yields of 4.1g/L,3.5g/L,0.76g/L,2.81g/L,8.2g/L and 6.85g/L, respectively, were detected from the fermentation supernatants by examination of NBC6-1 to NBC 6-6. N-acetylneuraminic acid (NeuAc) yields detected in NBC7-1 to NBC7-5 were 0.87g/L,0.765g/L,0.785g/L,0.905g/L and 0.8g/L, respectively.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of Jiangnan
<120> recombinant E.coli for increasing N-acetylneuraminic acid production
<130> BAA220674A
<160> 19
<170> PatentIn version 3.3
<210> 1
<211> 377
<212> PRT
<213> Neisseria meningitidis
<400> 1
Met Lys Arg Ile Leu Cys Ile Thr Gly Thr Arg Ala Asp Phe Gly Lys
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Tyr Lys Glu Val Thr Arg Glu Asn Tyr Gln His Thr Tyr Leu Phe Ser
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<210> 2
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<212> DNA
<213> artificial sequence
<400> 2
atgaaaagaa ttttatgcat cacaggaaca cgcgcagatt ttggcaaact gaaaccgctg 60
cttgcgtata ttgaaaatca tccggatctg gaacttcatt taatcgttac aggaatgcat 120
atgatgaaaa catacggcag aacatacaaa gaagtgacac gcgaaaacta ccaacataca 180
tacctgtttt caaaccaaat tcagggcgaa ccgatgggag cagtgctggg caacacaatc 240
acatttatct ctagactttc agatgaaatc gaaccggata tggtcatgat ccatggagat 300
agacttgaag cattagcggg agcagcggtg ggcgcgttat caagccgcct ggtctgtcat 360
attgaaggcg gagaattaag cggcacagtc gatgattcta ttcgccattc aatcagcaaa 420
cttagccata tccatctggt tgctaacgaa caagccgtta caagacttgt gcagatggga 480
gaaaaacgca aacatatcca tattatcggc tcaccggatt tagatgtgat ggcttcttca 540
acactgccga gccttgaaga agtcaaagaa tattatggac tgccgtacga aaactacggc 600
atctcaatgt ttcatccggt tacaacagaa gctcatctta tgccgcaata tgctgcccag 660
tattttaaag ccctggaact ttcaggacag aacattatca gcatttatcc gaataacgat 720
acaggcacag aaagcatcct tcaagaactg ctgaaatacc agagcgataa atttatcgct 780
tttccgtcta tcagatttga atattttctg gttcttctga aacatgccaa atttatggtg 840
ggaaatagct ctgctggcat tcgcgaagcc ccgctgtatg gagtcccgag catcgatgtt 900
ggcacaagac aatctaatcg ccatatggga aaatcaatca tccatacaga ttacgaaaca 960
aaaaacattt ttgatgcaat ccaacaggcg tgctctctgg gcaaatttga agcagatgat 1020
acatttaacg gcggagatac aagaacatct acagaacgct ttgcagaagt cattaataac 1080
ccggaaacat ggaatgtttc agcgcagaaa agatttatcg atttaaacct gtaa 1134
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<212> PRT
<213> Moritella viscosa
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Glu Ile Ile Lys His Gln Thr His Val Ile Glu Asp Glu Met Ser Ser
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Glu Ala Lys Lys Val Ile Pro Gly Asn Ala Asp Val Ser Ile Tyr Glu
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Lys Tyr Ile Glu Ser Lys Gly Ala Ile Phe Ile Ser Thr Pro Phe Ser
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Ile Gly Ser Gly Glu Cys Asn Asn Tyr Pro Leu Leu Asp Leu Ile Ala
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210 215 220
Val Ala Ala Gly Ala Ser Val Leu Glu Arg His Phe Thr Asp Asn Lys
225 230 235 240
Ala Arg Ser Gly Pro Asp Ile Cys Cys Ser Met Asp Gly Ala Glu Cys
245 250 255
Ala Glu Leu Ile Ser Gln Ser Lys Arg Met Ala Gln Met Arg Gly Gly
260 265 270
Ser Lys Gly Ala Val Lys Glu Glu Gln Val Thr Ile Asp Phe Ala Tyr
275 280 285
Ala Ser Val Val Thr Ile Lys Glu Ile Lys Ala Gly Glu Ala Phe Thr
290 295 300
Lys Asp Asn Leu Trp Val Lys Arg Pro Gly Thr Gly Asp Phe Leu Ala
305 310 315 320
Asp Asp Tyr Glu Met Leu Leu Gly Lys Lys Ala Ser Gln Asn Ile Asp
325 330 335
Phe Asp Val Gln Leu Lys Lys Glu Phe Ile Lys
340 345
<210> 4
<211> 1044
<212> DNA
<213> artificial sequence
<400> 4
atgacaaatc cggtctttga aatttctggc agaaaagttg gacttgatta tgccccgtta 60
gtgatcgcag aaattggcat caaccatgaa ggatcactga aaacagcctt tgaaatggtg 120
gatgcagcga ttgaaggcgg agcagaaatc atcaaacatc aaacacatgt cattgaagat 180
gaaatgtcaa gcgaagcaaa gaaagttatc ccgggcaatg ctgatgtgag catctacgaa 240
atcatggata gatgctctct gaacgaagaa gatgaaatca aactgaaaaa atacatcgaa 300
tcaaaaggcg ctatctttat ctcaacaccg tttagccgcg ctgccgcact gagacttgaa 360
cgcatgggag ttagcgccta taaaattggc tctggagaat gcaataacta tccgctgctt 420
gatcttattg cgtcttatgg caaaccggtc atcttatcaa caggaatgaa tgatattccg 480
tctatcagaa aatcagttga aatctttcgc aaatacaaaa caccgctttg tttactgcat 540
acaacaaacc tgtatccgac accggatcat cttattagaa tcggcgcaat ggaagaaatg 600
caacgcgaat ttagcgatgt tgtggtcgga ctgagcgatc attctatcga taacctggct 660
tgtctgggag ctgtggctgc tggagcttct gtcctggaaa gacattttac agataacaaa 720
gctcgctcag gcccggatat ttgctgtagc atggatggag cggaatgtgc tgaacttatc 780
tctcaatcaa aaagaatggc ccagatgcgc ggcggatcaa aaggcgcagt caaagaagaa 840
caggttacaa ttgattttgc ctatgcaagc gttgtgacaa ttaaagaaat caaagccgga 900
gaagcattta caaaagataa tctgtgggtt aaacgcccgg gcacaggaga ttttcttgcg 960
gatgattatg aaatgctttt aggcaagaaa gcaagccaaa acattgattt tgatgtgcag 1020
ctgaagaaag aatttatcaa ataa 1044
<210> 5
<211> 609
<212> PRT
<213> artificial sequence
<400> 5
Met Cys Gly Ile Val Gly Ala Ile Ala Gln Arg Asp Val Ala Lys Ile
1 5 10 15
Leu Leu Glu Gly Leu Arg Arg Leu Glu Tyr Arg Gly Tyr Asp Ser Ala
20 25 30
Gly Leu Ala Val Val Asp Ala Glu Gly His Met Thr Arg Leu Arg Arg
35 40 45
Leu Gly Lys Val Gln Met Leu Ala Gln Ala Ala Glu Glu His Pro Leu
50 55 60
His Gly Gly Thr Gly Ile Ala His Thr Arg Trp Ala Thr His Gly Glu
65 70 75 80
Pro Ser Glu Val Asn Ala His Pro His Val Ser Glu His Ile Val Val
85 90 95
Val His Asn Gly Ile Ile Glu Asn His Glu Pro Leu Arg Glu Glu Leu
100 105 110
Lys Ala Arg Gly Tyr Thr Phe Val Ser Glu Thr Asp Thr Glu Val Ile
115 120 125
Ala His Leu Val Asn Trp Glu Leu Lys Gln Gly Gly Thr Leu Arg Glu
130 135 140
Ala Val Leu Arg Ala Ile Pro Gln Leu Arg Gly Ala Tyr Gly Thr Val
145 150 155 160
Ile Met Asp Ser Arg His Pro Asp Thr Leu Leu Ala Ala Arg Ser Gly
165 170 175
Ser Pro Leu Val Ile Gly Leu Gly Met Gly Glu Asn Phe Ile Ala Ser
180 185 190
Asp Gln Leu Ala Leu Leu Pro Val Thr Arg Arg Phe Ile Phe Leu Glu
195 200 205
Glu Gly Asp Ile Ala Glu Ile Thr Arg Arg Ser Val Asn Ile Phe Asp
210 215 220
Lys Thr Gly Ala Glu Val Lys Arg Gln Asp Ile Glu Ser Asn Leu Gln
225 230 235 240
Tyr Asp Ala Gly Asp Lys Gly Ile Tyr Arg His Tyr Met Gln Lys Glu
245 250 255
Ile Tyr Glu Gln Pro Asn Ala Ile Lys Asn Thr Leu Thr Gly Arg Ile
260 265 270
Ser His Gly Gln Val Asp Leu Ser Glu Leu Gly Pro Asn Ala Asp Glu
275 280 285
Leu Leu Ser Lys Val Glu His Ile Gln Ile Leu Ala Cys Gly Thr Ser
290 295 300
Tyr Asn Ser Gly Met Val Ser Arg Tyr Trp Phe Glu Ser Leu Ala Gly
305 310 315 320
Ile Pro Cys Asp Val Glu Ile Ala Ser Glu Phe Arg Tyr Arg Lys Ser
325 330 335
Ala Val Arg Arg Asn Ser Leu Met Ile Thr Leu Ser Gln Ser Gly Glu
340 345 350
Thr Ala Asp Thr Leu Ala Gly Leu Arg Leu Ser Lys Glu Leu Gly Tyr
355 360 365
Leu Gly Ser Leu Ala Ile Cys Asn Val Pro Gly Ser Ser Leu Val Arg
370 375 380
Glu Ser Val Leu Ala Leu Met Thr Asn Ala Gly Thr Glu Ile Gly Val
385 390 395 400
Ala Ser Thr Lys Ala Phe Thr Thr Gln Leu Thr Val Leu Leu Met Leu
405 410 415
Val Ala Lys Leu Ser Arg Leu Lys Gly Leu Asp Ala Ser Ile Glu His
420 425 430
Asp Ile Val His Gly Leu Gln Ala Leu Pro Ser Arg Ile Glu Gln Met
435 440 445
Leu Pro Gln Asp Lys Arg Ile Glu Ala Leu Ala Glu Asp Phe Ser Asp
450 455 460
Lys His His Ala Leu Phe Leu Gly Arg Gly Asp Gln Tyr Pro Ile Ala
465 470 475 480
Leu Glu Gly Ala Leu Lys Leu Lys Glu Ile Ser Tyr Ile His Ala Glu
485 490 495
Ala Tyr Ala Ala Gly Glu Leu Lys His Gly Pro Leu Ala Leu Ile Asp
500 505 510
Ala Asp Met Pro Val Ile Val Val Ala Pro Asn Asn Gly Leu Leu Glu
515 520 525
Lys Leu Lys Ser Asn Ile Glu Glu Val Arg Ala Arg Gly Gly Gln Leu
530 535 540
Tyr Val Phe Ala Asp Gln Asp Ala Gly Phe Val Ser Ser Asp Asn Met
545 550 555 560
His Ile Ile Glu Met Pro His Val Glu Glu Val Ile Ala Pro Ile Phe
565 570 575
Tyr Thr Val Pro Leu Gln Leu Leu Ala Tyr His Val Ala Leu Ile Lys
580 585 590
Gly Thr Asp Val Asp Gln Pro Arg Asn Leu Ala Lys Ser Val Thr Val
595 600 605
Glu
<210> 6
<211> 1830
<212> DNA
<213> artificial sequence
<400> 6
atgtgtggaa ttgttggcgc gatcgcgcaa cgtgatgtag caaaaatcct tcttgaaggt 60
ttacgtcgtc tggaataccg cggatatgac tctgccggtc tggccgttgt tgatgcagaa 120
ggtcatatga cccgcctgcg tcgcctcggt aaagtccaga tgctggcaca ggcagcggaa 180
gaacatcctc tgcatggcgg cactggtatt gctcacactc gctgggcgac ccacggtgaa 240
ccttcagaag tgaatgcgca tccgcatgtt tctgaacaca ttgtggtggt gcataacggc 300
atcatcgaaa accatgaacc gctgcgtgaa gagctaaaag cgcgtggcta taccttcgtt 360
tctgaaaccg acaccgaagt gattgcccat ctggtgaact gggagctgaa acaaggcggg 420
actctgcgtg aggccgttct gcgtgctatc ccgcagctgc gtggtgcgta cggtacagtg 480
atcatggact cccgtcaccc ggataccctg ctggcggcac gttctggtag tccgctggtg 540
attggcctgg ggatgggcga aaactttatc gcttctgacc agctggcgct gttgccggtg 600
acccgtcgct ttatcttcct tgaagagggc gatattgcgg aaatcactcg ccgttcggta 660
aacatcttcg ataaaactgg cgcggaagta aaacgtcagg atatcgaatc caatctgcaa 720
tatgacgcgg gcgataaagg catttaccgt cactacatgc agaaagagat ctacgaacag 780
ccgaacgcga tcaaaaacac ccttaccgga cgcatcagcc acggtcaggt tgatttaagc 840
gagctgggac cgaacgccga cgaactgctg tcgaaggttg agcatattca gatcctcgcc 900
tgtggtactt cttataactc cggtatggtt tcccgctact ggtttgaatc gctagcaggt 960
attccgtgcg acgtcgaaat cgcctctgaa ttccgctatc gcaaatctgc cgtgcgtcgt 1020
aacagcctga tgatcacctt gtcacagtct ggcgaaaccg cggataccct ggctggcctg 1080
cgtctgtcga aagagctggg ttaccttggt tcactggcaa tctgtaacgt tccgggttct 1140
tctctggtgc gcgaatccgt tctggcgcta atgaccaacg cgggtacaga aatcggcgtg 1200
gcatccacta aagcattcac cactcagtta actgtgctgt tgatgctggt ggcgaagctg 1260
tctcgcctga aaggtctgga tgcctccatt gaacatgaca tcgtgcatgg tctgcaggcg 1320
ctgccgagcc gtattgagca gatgctgcct caggacaaac gcattgaagc gctggcagaa 1380
gatttctctg acaaacatca cgcgctgttc ctgggccgtg gcgatcagta cccaatcgcg 1440
ctggaaggcg cattgaagtt gaaagagatc tcttacattc acgctgaagc ctacgctgct 1500
ggcgaactga aacacggtcc gctggcgcta attgatgccg atatgccggt tattgttgtt 1560
gcaccgaaca acggattgct ggaaaaactg aaatccaaca ttgaagaagt tcgcgcgcgt 1620
ggcggtcagt tgtatgtctt cgccgatcag gatgcgggtt ttgtaagtag cgataacatg 1680
cacatcatcg agatgccgca tgtggaagag gtgattgcac cgatcttcta caccgttccg 1740
ctgcagctgc tggcttacca tgtcgcgctg atcaaaggca ccgacgttga ccagccgcgt 1800
aacctggcaa aatcggttac ggttgagtaa 1830
<210> 7
<211> 28
<212> DNA
<213> artificial sequence
<400> 7
ttgacaatta atcatcggct cgtataat 28
<210> 8
<211> 146
<212> DNA
<213> artificial sequence
<400> 8
attggctatc acatccgaca caaatgttgc catcccattg cttaatcgaa taaaaatcag 60
gctacatggg tgctaaatct ttaacgataa cgccattgag gctggtcatg gcgctcataa 120
atctggtata cttaccttta cacatt 146
<210> 9
<211> 160
<212> DNA
<213> artificial sequence
<400> 9
gcacaaaaaa tttttgcatc tcccccttga tgacgtggtt tacgacccca tttagtagtc 60
aaccgcagtg agtgagtctg caaaaaaatg aaattgggca gttgaaacca gacgtttcgc 120
ccctattaca gactcacaac cacatgatga ccgaatatat 160
<210> 10
<211> 88
<212> DNA
<213> artificial sequence
<400> 10
gattgatgac aatgtgagtg cttcccttga aaccctgaaa ctgatcccca taataagcga 60
agttagcgag atgaatgcga aaaaaacg 88
<210> 11
<211> 60
<212> DNA
<213> artificial sequence
<400> 11
aaaaaacggc ctctcgaaat agagggttga cactcttttg agaatatgtt atattatcag 60
<210> 12
<211> 80
<212> DNA
<213> artificial sequence
<400> 12
ttgaggaatc atagaatttt taatttaaat tttatttgac aaaaatgggc tcgtgttgta 60
taatctaagc tagtgtattt 80
<210> 13
<211> 82
<212> DNA
<213> artificial sequence
<400> 13
ttgaggaatc atagaatttt gacttaaaaa tttcagttgc ttaatcccta caattcttga 60
tataatattc tcatagtttg aa 82
<210> 14
<211> 30
<212> DNA
<213> artificial sequence
<400> 14
ttgacaatta atcatccggc tcgtataatg 30
<210> 15
<211> 191
<212> DNA
<213> artificial sequence
<400> 15
agcaacatct atcatctaaa aaaccagaaa aacaaataac atcatgtttt taaactaatt 60
aaatgaaata aaattttaag ccactcgcca ttgttcacaa taaaataaac tttataaatt 120
ttattttttt gtgaagtcgc cagcatcttt tctgttcttg ctgtggtgat atagtggcgt 180
cttcaattca a 191
<210> 16
<211> 349
<212> PRT
<213> Neisseria meningitidis
<400> 16
Met Gln Asn Asn Asn Glu Phe Lys Ile Gly Asn Arg Ser Val Gly Tyr
1 5 10 15
Asn His Glu Pro Leu Ile Ile Cys Glu Ile Gly Ile Asn His Glu Gly
20 25 30
Ser Leu Lys Thr Ala Phe Glu Met Val Asp Ala Ala Tyr Asn Ala Gly
35 40 45
Ala Glu Val Val Lys His Gln Thr His Ile Val Glu Asp Glu Met Ser
50 55 60
Asp Glu Ala Lys Gln Val Ile Pro Gly Asn Ala Asp Val Ser Ile Tyr
65 70 75 80
Glu Ile Met Glu Arg Cys Ala Leu Asn Glu Glu Asp Glu Ile Lys Leu
85 90 95
Lys Glu Tyr Val Glu Ser Lys Gly Met Ile Phe Ile Ser Thr Pro Phe
100 105 110
Ser Arg Ala Ala Ala Leu Arg Leu Gln Arg Met Asp Ile Pro Ala Tyr
115 120 125
Lys Ile Gly Ser Gly Glu Cys Asn Asn Tyr Pro Leu Ile Lys Leu Val
130 135 140
Ala Ser Phe Gly Lys Pro Ile Ile Leu Ser Thr Gly Met Asn Ser Ile
145 150 155 160
Glu Ser Ile Lys Lys Ser Val Glu Ile Ile Arg Glu Ala Gly Val Pro
165 170 175
Tyr Ala Leu Leu His Cys Thr Asn Ile Tyr Pro Thr Pro Tyr Glu Asp
180 185 190
Val Arg Leu Gly Gly Met Asn Asp Leu Ser Glu Ala Phe Pro Asp Ala
195 200 205
Ile Ile Gly Leu Ser Asp His Thr Leu Asp Asn Tyr Ala Cys Leu Gly
210 215 220
Ala Val Ala Leu Gly Gly Ser Ile Leu Glu Arg His Phe Thr Asp Arg
225 230 235 240
Met Asp Arg Pro Gly Pro Asp Ile Val Cys Ser Met Asn Pro Asp Thr
245 250 255
Phe Lys Glu Leu Lys Gln Gly Ala His Ala Leu Lys Leu Ala Arg Gly
260 265 270
Gly Lys Lys Asp Thr Ile Ile Ala Gly Glu Lys Pro Thr Lys Asp Phe
275 280 285
Ala Phe Ala Ser Val Val Ala Asp Lys Asp Ile Lys Lys Gly Glu Leu
290 295 300
Leu Ser Gly Asp Asn Leu Trp Val Lys Arg Pro Gly Asn Gly Asp Phe
305 310 315 320
Ser Val Asn Glu Tyr Glu Thr Leu Phe Gly Lys Val Ala Ala Cys Asn
325 330 335
Ile Arg Lys Gly Ala Gln Ile Lys Lys Thr Asp Ile Glu
340 345
<210> 17
<211> 1050
<212> DNA
<213> artificial sequence
<400> 17
atgcaaaaca acaacgaatt taaaatcggc aacagatcag tcggatataa tcatgaaccg 60
cttattatct gcgaaattgg catcaaccat gaaggaagct taaaaacagc ctttgaaatg 120
gtcgatgcag cgtataatgc cggagcagaa gttgtgaaac atcaaacaca tatcgttgaa 180
gatgaaatgt ctgatgaagc caaacaggtg atcccgggca acgcagatgt ctcaatctac 240
gaaatcatgg aaagatgtgc gctgaacgaa gaagatgaaa tcaaactgaa agaatacgtt 300
gaaagcaaag gaatgatctt tatctctaca ccgttttcac gcgctgccgc acttagatta 360
cagcgcatgg atattccggc ctataaaatc ggctctggag aatgcaacaa ctacccgctg 420
atcaaactgg tggcaagctt tggcaaaccg atcatcctgt ctacaggaat gaactcaatc 480
gaaagcatca aaaaatcagt tgaaatcatc agagaagcgg gcgtgccgta tgctctgctt 540
cattgtacaa acatttatcc gacaccgtat gaagatgttc gcctgggcgg aatgaatgat 600
ctttcagaag cctttccgga tgcaattatc ggccttagcg atcatacatt agataactat 660
gcatgcctgg gagcggtggc tcttggcgga tctatcctgg aaagacattt tacagataga 720
atggatcgcc cgggcccgga tatcgtctgt tcaatgaatc cggatacatt taaagaactg 780
aaacaaggag cccatgcact gaaacttgcg agaggcggca agaaagatac aattatcgct 840
ggcgaaaaac cgacaaaaga ttttgcgttt gctagcgtcg ttgcggataa agatattaag 900
aaaggcgaac tgctgtctgg agataacctg tgggtcaaaa gaccgggcaa cggagatttt 960
agcgttaacg aatacgaaac actttttggc aaagtggcgg cttgcaatat ccgcaaagga 1020
gctcagatta agaaaacaga tatcgaataa 1050
<210> 18
<211> 346
<212> PRT
<213> Escherichia coli
<400> 18
Met Ser Asn Ile Tyr Ile Val Ala Glu Ile Gly Cys Asn His Asn Gly
1 5 10 15
Ser Val Asp Ile Ala Arg Glu Met Ile Leu Lys Ala Lys Glu Ala Gly
20 25 30
Val Asn Ala Val Lys Phe Gln Thr Phe Lys Ala Asp Lys Leu Ile Ser
35 40 45
Ala Ile Ala Pro Lys Ala Glu Tyr Gln Ile Lys Asn Thr Gly Glu Leu
50 55 60
Glu Ser Gln Leu Glu Met Thr Lys Lys Leu Glu Met Lys Tyr Asp Asp
65 70 75 80
Tyr Leu His Leu Met Glu Tyr Ala Val Ser Leu Asn Leu Asp Val Phe
85 90 95
Ser Thr Pro Phe Asp Glu Asp Ser Ile Asp Phe Leu Ala Ser Leu Lys
100 105 110
Gln Lys Ile Trp Lys Ile Pro Ser Gly Glu Leu Leu Asn Leu Pro Tyr
115 120 125
Leu Glu Lys Ile Ala Lys Leu Pro Ile Pro Asp Lys Lys Ile Ile Ile
130 135 140
Ser Thr Gly Met Ala Thr Ile Asp Glu Ile Lys Gln Ser Val Ser Ile
145 150 155 160
Phe Ile Asn Asn Lys Val Pro Val Gly Asn Ile Thr Ile Leu His Cys
165 170 175
Asn Thr Glu Tyr Pro Thr Pro Phe Glu Asp Val Asn Leu Asn Ala Ile
180 185 190
Asn Asp Leu Lys Lys His Phe Pro Lys Asn Asn Ile Gly Phe Ser Asp
195 200 205
His Ser Ser Gly Phe Tyr Ala Ala Ile Ala Ala Val Pro Tyr Gly Ile
210 215 220
Thr Phe Ile Glu Lys His Phe Thr Leu Asp Lys Ser Met Ser Gly Pro
225 230 235 240
Asp His Leu Ala Ser Ile Glu Pro Asp Glu Leu Lys His Leu Cys Ile
245 250 255
Gly Val Arg Cys Val Glu Lys Ser Leu Gly Ser Asn Ser Lys Val Val
260 265 270
Thr Ala Ser Glu Arg Lys Asn Lys Ile Val Ala Arg Lys Ser Ile Ile
275 280 285
Ala Lys Thr Glu Ile Lys Lys Gly Glu Val Phe Ser Glu Lys Asn Ile
290 295 300
Thr Thr Lys Arg Pro Gly Asn Gly Ile Ser Pro Met Glu Trp Tyr Asn
305 310 315 320
Leu Leu Gly Lys Ile Ala Glu Gln Asp Phe Ile Pro Asp Glu Leu Ile
325 330 335
Ile His Ser Glu Phe Lys Asn Gln Gly Glu
340 345
<210> 19
<211> 1041
<212> DNA
<213> artificial sequence
<400> 19
atgtctaaca tctacatcgt ggcagaaatc ggctgcaatc ataacggatc agtcgatatc 60
gcgagagaaa tgattttaaa agctaaagaa gccggcgtga acgctgtcaa atttcaaaca 120
tttaaagccg ataaactgat cagcgcaatt gcgccgaaag cagaatacca aatcaaaaac 180
acaggagaat tagaatctca gctggaaatg acgaaaaaac tggaaatgaa atacgatgat 240
taccttcatc tgatggaata cgcagtcagc ctgaatcttg atgtttttag cacaccgttt 300
gatgaagatt ctattgattt tctggcgtca ctgaaacaaa aaatctggaa aattccgtca 360
ggcgaactgc ttaaccttcc gtacctggaa aaaatcgcta aacttccgat cccggataag 420
aaaattatca ttagcacagg catggccaca atcgatgaaa tcaaacagtc tgtctcaatc 480
tttatcaata acaaagtccc ggttggaaac atcacaatcc tgcattgtaa cacagaatat 540
ccgacaccgt ttgaagatgt taaccttaac gctatcaacg atctgaaaaa acattttccg 600
aaaaacaaca tcggcttttc tgatcattca agcggatttt atgcagcgat tgctgccgtt 660
ccgtatggca tcacatttat cgaaaaacat tttacactgg ataaaagcat gtctggaccg 720
gatcatcttg cttcaatcga accggatgaa ctgaaacatc tttgcattgg cgttagatgt 780
gtggaaaaat cactgggatc aaatagcaaa gttgtgacag ccagcgaaag aaaaaacaaa 840
atcgttgcac gcaaatctat catcgcgaaa acagaaatca aaaaaggaga agtgttttca 900
gagaaaaata tcacaacaaa aagaccgggc aacggaatta gcccgatgga atggtataat 960
ttactgggca aaatcgcgga acaagatttt atcccggatg aacttatcat ccatagcgaa 1020
tttaaaaacc agggagaata a 1041

Claims (5)

1. A recombinant E.coli is characterized in that genes related to a glucosamine transport-related phosphotransferase system, such as N-acetylglucosamine-6-phosphodeacetylase nagA, glucosamine-6-phosphodeaminase nagB, N-acetylglucosamine specific EIICBA component nagE, N-acetylneuraminic acid transport carrier-related genes, such as N-acetylneuraminic acid lyase nanA, sialic acid transporter nanT, N-acetylmannosamine-6-phospho2-epimerase nanE, N-acetylmannosamine kinase nanK, mannose-specific EIIAB component-related genes manXYZ and pyruvate oxidase poxB genes in genome are knocked out, and endogenous enzyme glmM regulated by low-strength constitutive promoters, glmU regulated by medium-strength constitutive promoters and glucose amine synthase mutant glmS are expressed A The method comprises the steps of carrying out a first treatment on the surface of the The low-strength constitutive promoter is P grpE The medium-strength constitutive promoter is P ssrA The method comprises the steps of carrying out a first treatment on the surface of the The glmM, glmU and the glucosamine synthase mutant glmS A The integration site of the gene is the position of the motA gene of the escherichia coli; and incorporate P at ΔmanXYZ grpE Regulated UDP-N-acetylglucosamine-2-epimerase Gene NeuC and integration of P at ΔpoxB grpE A regulated neisseria meningitidis (Neisseria meningitidis) derived N-acetylneuraminic acid synthase gene nemaneub;
the nucleotide sequence of the Gene nagA is shown as Gene ID 945289, the nucleotide sequence of the Gene nagB is shown as Gene ID 945290, and the nucleotide sequence of the Gene nagE is shown as Gene ID 945292; the nucleotide sequence of the Gene nanA is shown in Gene ID 947742); the nucleotide sequence of the Gene nanT is shown as Gene ID 947740; the nucleotide sequence of the Gene nanE is shown as Gene ID 947745; the nucleotide sequence of the Gene nanK is shown as Gene ID 947757; gene poxBThe nucleotide sequence is shown as Gene ID 946132; the nucleotide sequence of glmU is shown as Gene ID 948246; the nucleotide sequence of glmM is shown as Gene ID 947692; the glucosamine synthase mutant glmS A The nucleotide sequence of the gene of (2) is shown as SEQ ID NO. 6; the nucleotide sequence of the N-acetylneuraminic acid synthase NemNauB is shown as SEQ ID NO. 17; the promoter P ssrA The nucleotide sequence of (2) is shown as SEQ ID NO. 8; the P is grpE The nucleotide sequence of (2) is shown as SEQ ID NO. 10; the nucleotide sequence of the coding NeuC gene is shown as SEQ ID NO. 2.
2. A method for improving the synthesis capability of recombinant escherichia coli N-acetylneuraminic acid is characterized in that based on an initial strain, genes N-acetylglucosamine-6-phosphate deacetylase nagA, glucosamine-6-phosphate deaminase nagB, N-acetylglucosamine specific EIICBA component nagE, N-acetylneuraminic acid transport vector related genes N-acetylneuraminic acid lyase nanA, sialic acid transport protein nanT, N-acetylmannosamine-6-phosphate 2-epimerase nanE, N-acetylmannosamine kinase nanK, mannose specific EIIAB component related genes manXZ and pyruvate oxidase poxB genes in a genome are knocked out, and endogenous enzyme glmM regulated by low-strength constitutive promoters, glmU regulated by medium-strength constitutive promoters and glucosamine synthase mutant glmS are expressed A The method comprises the steps of carrying out a first treatment on the surface of the The low-strength constitutive promoter is P grpE The medium-strength constitutive promoter is P ssrA The method comprises the steps of carrying out a first treatment on the surface of the The glmM, glmU and the glucosamine synthase mutant glmS A The integration site of the gene is the position of the motA gene of the escherichia coli; and incorporate P at ΔmanXYZ grpE Regulated UDP-N-acetylglucosamine-2-epimerase Gene NeuC and integration of P at ΔpoxB grpE A regulated neisseria meningitidis (Neisseria meningitidis) derived N-acetylneuraminic acid synthase gene nemaneub;
the nucleotide sequence of the Gene nagA is shown as Gene ID 945289, the nucleotide sequence of the Gene nagB is shown as Gene ID 945290, and the nucleotide sequence of the Gene nagE is shown as Gene ID 945292The method comprises the steps of carrying out a first treatment on the surface of the The nucleotide sequence of the Gene nanA is shown in Gene ID 947742); the nucleotide sequence of the Gene nanT is shown as Gene ID 947740; the nucleotide sequence of the Gene nanE is shown as Gene ID 947745; the nucleotide sequence of the Gene nanK is shown as Gene ID 947757; the nucleotide sequence of the Gene poxB is shown as Gene ID 946132; the nucleotide sequence of glmU is shown as Gene ID 948246; the nucleotide sequence of glmM is shown as Gene ID 947692; the glucosamine synthase mutant glmS A The nucleotide sequence of the gene of (2) is shown as SEQ ID NO. 6; the nucleotide sequence of the N-acetylneuraminic acid synthase NemNauB is shown as SEQ ID NO. 17; the promoter P ssrA The nucleotide sequence of (2) is shown as SEQ ID NO. 8; the P is grpE The nucleotide sequence of (2) is shown as SEQ ID NO. 10; the nucleotide sequence of the coding NeuC gene is shown as SEQ ID NO. 2.
3. A method for producing N-acetylneuraminic acid, which comprises fermenting the recombinant E.coli according to claim 1.
4. A method according to claim 3, wherein the fermentation is carried out at 35-37 ℃ for at least 24 hours using glycerol as a carbon source.
5. Use of the escherichia coli of claim 1, or the method of any one of claims 3 to 4, for the production of a product comprising N-acetylneuraminic acid.
CN202210690425.4A 2022-06-17 2022-06-17 Recombinant escherichia coli for improving yield of N-acetylneuraminic acid Active CN114891712B (en)

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CN109971696A (en) * 2019-03-20 2019-07-05 江南大学 A kind of recombinant bacterium of resting cell method high yield N-acetyl-neuraminate and application
CN113122491A (en) * 2021-03-26 2021-07-16 清华大学 Recombinant microorganism for producing N-acetylneuraminic acid and application thereof
CN113817658A (en) * 2021-08-24 2021-12-21 天津科技大学 Genetically engineered bacterium for producing N-acetylneuraminic acid and construction and application thereof

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CN109971696A (en) * 2019-03-20 2019-07-05 江南大学 A kind of recombinant bacterium of resting cell method high yield N-acetyl-neuraminate and application
CN113122491A (en) * 2021-03-26 2021-07-16 清华大学 Recombinant microorganism for producing N-acetylneuraminic acid and application thereof
CN113817658A (en) * 2021-08-24 2021-12-21 天津科技大学 Genetically engineered bacterium for producing N-acetylneuraminic acid and construction and application thereof

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